Abstract: Melanoma is the most deadly form of skin cancer, characterized by aggressive clinical behavior and propensity for lethal metastasis. Melanoma is driven by mutational activation of BRAFV600E accompanied by PTEN loss resulting in concurrent activation of BRAF/MEK/ERK (MAPK) and the PI3K/AKT/mTOR (PI3K) pathways. Activation of these pathways plays crucial role in melanoma cell proliferation, survival, tumorigenesis, invasion and metastasis. Agents that target the MAPK signaling pathway, the BRAF inhibitors (vemurafenib and dabrafenib), and the MEK inhibitor (trametinib) in combination with dabrafenib, have shown remarkable anti- tumor activity and improved the overall survival of patients with metastatic melanoma. However, most patients still developed resistance and ultimately succumbed to metastatic disease. In the majority of cases, resistance to BRAF and MEK inhibitors occurs through reactivation of MAPK pathway and activation of the PI3K signaling. This paradigm of chemoresistance with scarce treatment alternatives signals an urgent need to develop novel agents that down-regulate PI3K pathway to achieve durable clinical control of BRAFV600E- mutated melanoma along with BRAF inhibitors. Thus, identification of phytochemicals that can be used in combination with lower doses of existing chemotherapeutic drugs is of high clinical relevance. Plumbagin (5- hydroxy-2-methyl-1,4-naphthoquinone), a naturally occurring naphthoquinone isolated from the plant Plumbago zeylanica L., exhibits anti-proliferative and anti-tumorigenic activities. Consistent with this notion, our preliminary findings supported by this grant application have helped us begin to define the utility of plumbagin for the treatment of melanoma. Our data indicate that plumbagin treatment induces apoptosis, and reduces the proliferation, migration, and invasion of BRAFV600E-mutated melanoma cells. Plumbagin's effects resulted from decreased expression of PI3K and reduced phosphorylation of AKT. In this application, we propose to capitalize on our novel findings to investigate the effects of plumbagin (that targets PI3K/AKT) in combination with vemurafenib (a BRAF inhibitor) on the proliferation, tumorigenesis and metastasis of BRAFV600E-mutated melanoma cells. We hypothesize that the treatment of BRAFV600E-mutated melanoma cells with plumbagin could offer a therapeutic advantage by inhibiting PI3K/AKT in combination with vemurafenib over an individual agent. We propose a two-fold approach to test this hypothesis. First, we will investigate the effects of plumbagin and vemurafenib alone and in combination on proliferation, apoptosis and invasion of BRAFV600E- mutated melanoma cells in vitro and in three-dimensional human melanoma skin eqivalents. Second, we will investigate the effects of plumbagin and vemurafenib alone and in combination on tumor growth, metastasis and survival by employing a genetically engineered mouse model of melanoma. Together, these studies will likely provide novel treatment options for this life threatening skin cancer.